Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7871-7875, November 1987 Biochemistry A DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression (steroid hormone action/steroid receptor/transcription regulation) U. STRAHLE, G. KLOCK, AND G. SCHUTZ Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany Communicated by Elwood V. Jensen, July 27, 1987 ABSTRACT To defmie the recognition sequence of the the complementary strands, which generated BamHI or Xba glucocorticoid receptor and its relationship with that of the I cohesive ends, and cloning upstream of the herpes simplex progesterone receptor, oligonucleotides derived from the glu- virus thymidine kinase (TK) promoter in pBLCAT2 (10) were cocorticoid response element of the tyrosine aminotransferase done by standard procedures (11). pBLCAT2 contains the gene were tested upstream of a heterologous promoter for their complete TK promoter (-105 to +62) linked to the chlor- capacity to mediate effects of these two steroids. We show that amphenicol acetyltransferase (CAT) coding region (12). In a 15-base-pair sequence with partial symmetry is sufficient to the plasmids pGRE22, pGREPal, and pGRE16, the binding confer glucocorticoid inducibility on the promoter ofthe herpes sites were synthesized as 27-bp oligonucleotides that were simplex virus thymidine kinase gene. The same 15-base-pair inserted into the BamHI site of the polylinker immediately sequence mediates induction by progesterone. Point mutations upstream ofthe TK promoter in pBLCAT2. In pGRE15A and in the recognition sequence affect inducibility by glucocorti- pGRE15B and in the plasmids containing single or double coids and progesterone similarly. Together with the strong point mutations, the binding sites were synthesized as 21-bp conservation ofthe sequence ofthe DNA-binding domain ofthe oligonucleotides and cloned into the Xba I site of the two receptors, these data suggest that both proteins recognize polylinker. By inserting the 21-bp oligonucleotides into the a sequence that is similar, if not the same. Xba I site and the 27-bp oligonucleotides into the BamHI site ofthe polylinker, the centers ofthe pentadecamer recognition The effect of steroid hormones on specific gene transcription sequences were put into the same position relative to the TK is mediated by receptors to which the hormones bind with promoter in all the plasmid constructs. high selectivity and affinity. Activation of transcription Cell Culture and Transfection. MCF7 and T-47D human results from binding of the hormone-receptor complex to breast cancer cells were cultured as described (4). Transfec- specific sequences of inducible genes (1, 2). Surprisingly, it tion of MCF7 and T-47D cells by the DEAE-dextran proce- has been observed that fragments of the chicken lysozyme dure was performed as described (4), with two modifications: gene and of the long terminal repeat of mouse mammary (i) the plasmid concentration in the DEAE-dextran mixture tumor virus that confer glucocorticoid inducibility also allow was reduced to 1 ,ug/ml and (ii) after treatment with chloro- progesterone induction (3, 4). The glucocorticoid and pro- quine diphosphate, the cells were kept in Dulbecco's modi- gesterone receptors bind to similar sequences within these fied Eagle's medium (DMEM) supplemented with 10% char- fragments, suggesting that the binding sites may be closely coal-stripped fetal bovine serum. After 14-18 hr the medium related, but distinct (5, 6). Both types of receptors also bind was replaced by serum-free DMEM and the incubation was to partially overlapping sequences in the rabbit uteroglobin continued for another 12-14 hr before harvesting. Immedi- gene (7, 8). These sequences, however, have not been ately after chloroquine diphosphate treatment, hormone functionally characterized. To define the sequence require- alone or hormone and antagonist were administered in ments for specific binding of the glucocorticoid receptor and ethanol solution to a final concentration of 0.1% ethanol. to determine the degree to which this sequence overlaps with Unless otherwise stated, 0.1 ,uM dexamethasone or 0.1 ,uM the recognition sequence for the progesterone receptor, a R 5020 (a synthetic gestagen; ref. 4) was applied (see also minimal glucocorticoid response element (GRE) 15 base pairs legends to figures). Control plates contained 0.1% ethanol. (bp) in length was defined. This 15-bp sequence is part of the CAT Assays. CAT assays were performed as described GRE region of the tyrosine aminotransferase (TAT) gene (12). After exposure to x-ray film, radioactive spots on the protected against DNase I digestion by the purified gluco- thin-layer chromatogram were cut out for quantitation by corticoid receptor (9). We demonstrate here that the same liquid scintillation counting. All numbers given, with the 15-mer which confers responsiveness to glucocorticoids also exception ofthe data for the dose-response curves, represent mediates progesterone induction. Since point mutations in the average of CAT activity (pmol per min per mg of protein) this 15-bp sequence affect inducibility by both steroids from two parallel transfections. similarly, we suggest that the recognition sequences of the RNA 5'-End Mapping. Total RNA was prepared by ex- glucocorticoid and progesterone receptors are similar, if not traction with guanidinium thiocyanate followed by centrifu- identical. gation through a CsCl cushion (13). Transcription start sites were determined by the RNase-protection procedure, using MATERIALS AND METHODS uniformly labeled antisense RNA probes (4, 14, 15). Twenty- microgram aliquots of total cellular RNA were hybridized Plasmid Constructions. Oligonucleotides were prepared with the probe and further processed as described elsewhere with an Applied Biosystems 380A DNA synthesizer and (4), with the modification that the concentration ofRNase T1 purified as recommended by the manufacturer. Annealing of was reduced to 20 units/ml. The publication costs of this article were defrayed in part by page charge Abbreviations: GRE, glucocorticoid-response element; CAT, chlor- payment. This article must therefore be hereby marked "advertisement" amphenicol acetyltransferase; TAT, tyrosine aminotransferase; TK, in accordance with 18 U.S.C. §1734 solely to indicate this fact. thymidine kinase. 7871 Downloaded by guest on September 25, 2021 7872 Biochemistry: Strahile et al. Proc. Natl. Acad. Sci. USA 84 (1987) RESULTS gene is sufficient to confer glucocorticoid inducibility on the TK promoter in MCF7 cells. This oligonucleotide corre- The Glucocorticoid Receptor Binding Site Is Sufficient to sponds to the sequence that is protected by the purified Confer Inducibility on a Transcriptionaily Competent Promot- glucocorticoid receptor against digestion by DNase I and er. To define the sequence required to constitute a GRE, we placed oligonucleotides modeled after a receptor binding site displays strong similarity with the GREs of other glucocor- of the TAT gene (9) upstream of the TK promoter. The ticoid-inducible genes (9). A characteristic ofthis sequence is promoter activity was followed in recombinant plasmids in a 15-bp motif with partial dyad symmetry (indicated by which this promoter was fused to the bacterial CAT gene (12). arrows in Fig. 1). We also tested whether the symmetrical Expression of these plasmids was studied after transient portion ofthis sequence alone is capable ofrendering the TK introduction into the human breast cancer cell lines MCF7 promoter glucocorticoid-inducible. The 22-bp oligonucleo- and T-47D, which have glucocorticoid and progesterone tide in which most of the nucleotides outside the partial receptors (16), respectively. Glucocorticoid- and progester- palindrome have been modified (pGRE16) maintains the one-dependent expression ofthese plasmids was analyzed by capacity to make the TK promoter glucocorticoid-inducible following CAT enzymatic activity (Figs. 1, 3, and 4) and by (Fig. 1A). To test whether the GRE consensus sequence (9) measuring TK-CAT mRNA levels (Fig. 2) with a RNase- itself is sufficient for glucocorticoid control, a 15-bp-long protection assay (14, 15). sequence was examined. The oligomer comprising only 15 bp As seen in Fig. 1A, a 22-bp fragment derived from the TAT of GREII (pGRE15A) of the TAT gene leads to a strong A CAT Activity (pnoloslmin-mg) 100 200 300 -105 DEX I + pBLCAT2 x B + 4 GATCCTGTACAGGATGTTCTAGCTACG pGRE22 GACATGTCCTACAAGATCGATGCCTAG 4 pGREPaI GATCCAGAACATGATGTTCTAGCTACG 41 GTCTTGTACTACAAGATCGATGCCTAG +1 0 + pGRElS GATCCTGTACAGGATGTTCTCTAGCGC GACATGTCCTACAAGAGATCGCGCTAG +I pGREISA CTAGATGTACAGGATGTTCTT TACATGTCCTACAAGAAGATC . + U~ PGRE15B CTAGAAGAACATCCTGTACAT .1 TTCTTGTAGGACATGTAGATC Ir B CAT Activity (pmoles/min* mg) 50 100 -105 R55020 150 4+ pBLCAT2 x a 4+ GATCC TG TACAGGATGTTC TAGC TACG r1 pGRE22 GACATGTCC TACAAGATCGATGCC TAG GATCCAGAACATGATGTTCTAGCTACG pGREPal 'I. GTCTTGTACTACAAGATCGATGCCTAG + w C pGRE SA TAGATGTACAGGATGTTCTT 4. TACATG1 CCTACAAGAAGATC +w FIG. 1. Induction of CAT expression of TK-CAT constructs containing GREs by dexamethasone and R 5020 in MCF7 (A) and T-47D (B) cells. The oligonucleotides were inserted upstream ofthe TK-CAT fusion gene in pBLCAT2. After transient introduction into MCF7 and T-47D by the DEAE-dextran procedure, CAT activity was measured without hormone added (-) or in the presence (+) of 0.1 ,uM dexamethasone (A) or 0.1 ,uM R 5020 (B).